Title: p24 revisited: A landscape review of antigen detection for early HIV diagnosis

Supplemental Digital Information

Eleanor R GRAY1, Robert BAIN2, Olivia VARSANEUX3, Rosanna W PEELING3, Molly M STEVENS2, Rachel A
MCKENDRY*1,4
Supplementary Table 1

Detection time relative to

Study Assays used for early detection Reference test
reference test

Hashida Immune complex transfer EIA (for Ab ELISA, agglutination test

Ag - 7-21 days before
1996 [1] and Ag) (for Ab), and Western blot

Sickinger Combined-format HIV antigen-antibody

6.15 days before 3rd generation Ab tests
2004 [2] (AxSYM HIV Ag/Ab Combo)

3.6 to 5.7 days before 3rd generation Ab tests

Weber 2002 Enzyme immunoassay
[3] (Cobas Core HIV Combi EIA)
2.75 days after RT-PCR

Fifteen 4th generation assays

(VIDAS DUO Ultra to Vironostika HIV 2.4-6.8 days after PCR
uniform II Ag: Ab)
Weber 2006
[4]
Two 3rd generation assays
(Genscreen HIV 1/2, v2 to Ortho HIV- 6.15-10.25 days after PCR
1/HIV-2 Ab capture)

Supplementary Table 1 Summary of the timing of detection of different assays to detect HIV in comparison to
reference and p24 assays. Ag – antigen, Ab – antibody.
Supplementary Table 2

Name Manufacturer Applicability Sensitivity (%) Specificity (%) References

AxSYM Combo Abbott Laboratories Laboratory 99.6-100 98.0-100 [2,5-11]

Architect HIV Abbott Laboratories Laboratory 99.9-100 99.5-100 [6-8,12-14]

VIDAS HIV
DUO bioMerieux Laboratory 95.3-100 98.1-100 [3,5,10,11,15,16]
Ultra/Quick

LG HIV Ag–Ab
LG Laboratory 100 99.9 [17]
ELISA PLUS

Elecsys Combi Roche Laboratory 100 99.8-99.9 [7,8,12,18]

Elecsys HIV
Roche Laboratory 100 99.8-100 [6,8,12,18]
Combi PT

Enzygnost HIV
Siemens Laboratory 100 99.2-100 [3,5,16,19]
Integral

GS HIV Combo
Siemens Laboratory 100 96.7-100 [9,20,21]
Ag/Ab EIA

Genscreen Ultra
Bio-Rad Laboratory 99.7-100 98.1-100 [3,11,16,22]
HIV Ag/Ab

Vironostika HIV-
1 p24 antigen bioMerieux Laboratory 91.1-100 96.7-100 [11,12,15,16,19]
assay

BioPlex 2200
HIV Ag-Ab Bio-Rad Laboratory 100 99.56 [23]
assay

Acute infection
(Ag+/Ab-; Ag+/Ab+)

Ag+: 0-82.1
0-100
Alere Determine Ab+: 0-66.7
Alere PoC/Lab [24-36]
HIV-1/2 Ag/Ab
Established infection
(Ag+/Ab+; Ag-/Ab+)

88.2-99.8 89.5-100

Supplementary Table 2: Commercial assays for p24, with a summary of sensitivity and specificity derived
from published studies. Of the assays listed, only the BioPlex 2200 and Alere Determine give separate
readouts for antigen and antibody results. Only studies from independent published trials are included.
See Supplementary Table 3 for a breakdown of results from each study (including subsets that included
samples from acute infection).
Supplementary Table 3

Sample Sensitivity§ Specificity§ N

Reference Index Test Reference method
type(s) (%) (%) (samples)

Infants <14
days, 83 94 23
plasma, USA HIV culture, PCR
HIV Ag from PBMC,
Infants 16
ELISA with repeated Ab+ve at
Chandwani days-2
ICD*1 >15 months, or 60 100 13
1993 [37] months,
(Beckman seroreversion
plasma, USA
Coulter) (detection method
Infants 2-5 not stated)
months, 100 100 9
plasma, USA

HIV Ab assay
HIV-1 p24
Children <10 (Organon-Teknika) at 404
Quinn 1993 ELISA
yrs, serum, >15 months of age 89.5 99.2 (158
[38] (Coulter) with
USA with symptom patients)
ICD
assessment

Infants 1 HIV Ag
Bredberg- 53.0-70.8
week-1.5 yrs, ELISA Nested PCR (in-
Rådén (age 100 69
PBMC/plasm (Coulter) with house)
1995 [39] stratified)
a, Tanzania ICD

Infants 6
Western blot at ≥12
weeks and 3 HIV-1 p24 Ag 220
Bulterys months (Bio-Rad)
months, assay with 15.4 100 (36
1995 [40] and symptom
plasma, ICD (Abbott) patients)
assessment
Rwanda

Ag ELISA
(Coulter) with 58 85
ICD
Infants <3
Lewis 1995 months, cord NIAID ELISA and Western
46
[41] blood or protocol HIV blot at 15 months 83 100
plasma, USA culture

In-house
83 94
DNA PCR
 With ICD –
Infants, <5 HIV p24 EIA
Nielsen ELISA & Western 71.4
yrs, plasma, (Abbott) with 95 40
1995 [42] blot > 18 months No ICD –
Brazil ICD
52.4

HIVChek (DuPont),
Ag ELISA
Ab HIV-1 ELISA
Infants/adults (DuPont)
(Wellcozyme,
Lyamuya , with ICD and 85.8 1st test, 507 (453
Murex), Western blot 97.6
1996 [43] plasma/seru ELAST 100 overall patients)
(Diagnostic
m, Tanzania enhancemen
Biotechnology) or
t
PCR (in-house)

Infants 1 wk- Infected: Ab +ve at

HIV p24
1 yr, 18 months
Nesheim ELISA 35-93.3 (age 93-100 (age
PBMC/plasm Uninfected: 2x Ab - 345
1997 [44] (Coulter) with stratified) stratified)
a/serum, ve at >6 months
ICD
USA PCR on PBMC

Infants 1 Ab by ELISA & With ICD –

Panakitsuw Ag EIA
day-4 yrs, gelatin particle 85.4/87.8
an 1997 (Coulter) with 100 71
serum, agglutination, RT- No ICD –
[45] ICD
Thailand PCR 34.2

Infants <4 HIV-1 p24

HIV culture and 253
Paul 1997 months, ELISA
symptom 59 100 (206
[46] plasma/seru (Coulter) with
assessment patients)
m, USA ICD

90 (<7 days)
Infants, 0-6 HIV-1 p24 27 (<7 days)
100 (1
Rich 1997 months, ELISA HIV culture, ≥ 2 77 (1 month) 207
month)
[47] plasma/seru (Coulter) with positive results 71-81 (1 to 6 patients
98-100 (1 to
m, USA ICD months)
6 months)

NAAT (Amplicor,
Roche or bDNA,
Adults,
Daar 2001 HIV p24 Ag Chiron), Western blot
primary HIV, 88.7 100 436
[48] EIA, (Abbott) (Cambridge Biotech
plasma, USA
Corp.), or EIA
(Abbott)

Hecht 2002 HIV p24 Ag Genscreen HIV Ultra

79 99.5 258
[49] EIA (Abbott) (Bio-Rad), VIDAS
 bDNA Duo Ultra
100 95.3
(Bayer) (bioMérieux), VIDAS
Duo Quick
Amplicor
(bioMérieux),
PCR, 100 97.3
Architect HIV Combo
(Roche)
Adults, (Abbott), Elecsys HIV
primary HIV, Combi (Roche);
TMA HIV-1
plasma, USA Genscreen HIV v2
RNA, 100 98.4
(GenProbe) (Bio-Rad), Ortho HIV
Capture (Ortho
Ab-test (Ag Diagnostic Systems)
Combi EIA, 77 96.8
Abbott)

Cobas HIV
combi EIA 100 99.7
(Roche)

VIDAS HIV
DUO 100 100
(bioMérieux)

Enzygnost
HIV Integral
100 99.7
(Dade
Adults, blood
Behring)
donor >11,000
panels, Enzymun- total across
Weber plasma/seru Test HIV all sites, not
Various*2 100 100
2002 [5] m/culture Combi all samples
supernatants (Boehringer) tested on
, multi- all assays
Genscreen
country.
HIV1/2,
98.3 100
version 2
(Bio-Rad)

IMx HIV-
1/HIV-2 III 98.7 ND
Plus (Abbott)

AxSYM HIV-
1/2 gO 100 ND
(Abbott)
 Prism Anti-
HIV1/2 ND 99.9
(Abbott)

Enzygnost
Anti-HIV1/2
99.8 99.9
plus (Dade
Behring)

Cobas Core
Anti-HIV 1 2
98.3 ND
O EIA
(Roche)

Vironostika
Infants 1-6
HIV Ag/Ab 471
Sutthent months, NAAT (Amplicor
(bioMérieux) 100 100 (391
2003 [50] plasma/seru PCR, Roche)
with ICD and patients)
m, Thailand
ELAST

Worldwide
subtype 2.5 to
- 31
panel, >25pg/ml
supernatant
HIV-1 Ag MAb assay,
Worldwide 6 combined HIV-1 p24 Ag
Ly 2004 samples, Ag/Ab tests, quantitation assay 669+ve,
99.8-100 97.2-100
[11] plasma/seru and 2 Ab- (both Abbott) or 1005 -ve
m only tests*3 Cobas core HIV Ag
EIA (Roche)
176 from
Seroconversi
25
on panels, 38.1-55.1 -
seroconver
plasma
sion panels

HIV-1 p24
ELISA
(Perkin
Infants <1yr, 203
Sherman Elmer) with NAAT (Amplicor
plasma, 98.1 98.7 (90
2004 [51] ICD and PCR, Roche)
South Africa patients)
ELAST
enhancemen
t

Sickinger Adults, AxSYM HIV Western blot (LAV-

100 (Ab) 99.87-99.92 9838
2004 [2] Panels, Ag/Ab blot I & II, Bio-Rad),
 plasma/seru Combo HIV Ag Confirmatory 17.5pg/ml
m, (Abbott) (Murex), NAAT (Ag)
multicentre (Monitor, Roche)

HIV-1 p24
ELISA 46.4
Adult, B- 451
Respess (Coulter) with Versant bDNA assay (VL<30,000)
subtype, 100 (284
2005 [52] ELAST (Bayer) 100 (VL
plasma, USA patients)
enhancemen >30,000)
t

HIV Ag
ELISA
Infants 1-24
(Perkin 169
Nouhin months, DNA PCR (in-house)
Elmer) with 91.3 100 (147
2006 [53] plasma, and viral cultures
ICD and patients)
Cambodia
ELAST
amplification

Infants 1 HIV p24 Ag NAAT (DNA PCR

Patton month-12 ELISA Amplicor, Roche, or
98.8 100 141
2006 [54] yrs, DBS, (Perkin RNA NucliSens,
South Africa Elmer) bioMérieux)

HIV-positive LG HIV
& Ag/Ab Plus Enzygnost HIV
Yeom 2006
seroconversi ELISA (LG Integral (Dade 100 99.9-100 1282
[17]
on panels, Life Behring)
serum. Sciences)

HIV p24 Ag
ELISA
Infants <180 (Perkin NAAT (Roche 802
Fiscus
days, Elmer) with Amplicor HIV DNA) 91.7 98.5 (582
2007 [55]
plasma, USA ELAST and/or HIV culture patients)
enhancemen
t

Vironostika
HIV Ag/Ab
Infants 91 97
ELISA NAAT (RNA, 401
George IQR: 0.2 to
(bioMérieux) NucliSENS EasyQ (233
2007 [15] 3.6 yrs,
HIV-1, bioMérieux) patients)
plasma, Haiti
Vironostika
93 99
HIV Ag/Ab
 ELISA with
ELAST
enhancemen
t

VIDAS Duo
HIV Ag/Ab 95 99
(bioMérieux)

11 (6)
combined
Ly 2007 Ag/Ab tests;
See [11] See [11] 99.4-100 97.2-100 1983
[56] 2 (2) Ab-only
tests (from
[11]). *3

HIV Ag
ELISA NAAT (DNA PCR,
Infants 20
(Perkin Amplicor, Roche, or
Patton days-6 yrs,
Elmer) with RNA, NucliSENS 88.9-98.3 100 246
2008 [57] DBS, South
ELAST EasyQ HIV-1,
Africa
enhancemen bioMérieux)
t

DBS <20
months old:
HIV Ag
94.4 (50-100, 502
ELISA
NAAT (DNA PCR, age
(Perkin
Amplicor (Roche) or stratified)
Cachafeiro Infants, DBS, Elmer) with
in-house, or RNA 100
2009 [58] pan-country ICD and
NucliSens QT, DBS >20
ELAST
bioMerieux) months old:
enhancemen
72.2 (65.6- 115
t
100, age
stratified)

Alere
Determine 86.6
Panels, Ab/Ag
plasma/seru
Beelaert
m/whole Vironostika Various*4 100 379
2010 [31]
blood/culture HIV Uni-

supernatant Form II 92.5

Ag/Ab
(bioMérieux)
 HIV-1 p24
ELISA
Mwapasa Infants 6wks, HIV-1 DNA PCR
(Perkin 84 98 222
2010 [59] DBS, Malawi (Roche)
Elmer) with
ICD

NAAT
(Cavidi
274 (with
ExaVir), HIV-
modificatio
1 p24 ELISA
75 with 54 with n) or 306
(Perkin NAAT (Amplicor
Stewart Adults, modification, modification (without
Elmer) with Monitor HIV RNA
2010 [60] plasma, USA 66 without 44 without modificatio
ICD and PCR Roche)
modification modification n)
ELAST
(108
enhancemen
patients)
t +/-
modification

Adult
Alere RT Advance Quality 99.8 1019
Bhowan women, 100 (plasma
Determine HIV (Intec), Acon (plasma) (plasma) +
2011 plasma/whol and whole
Ag/Ab HIV-1/2/O Tri-line 99.3 (whole 380 (whole
[61] e blood, blood)
(Inverness) (Acon) blood) blood)
South Africa

EIA for Ag/Ab

Stored Alere
Fox 2011 (VIDAS Duo,
serum, p24 Determine 50.0 N/A 36
[34] bioMérieux) or EIA
Ag+ve, UK Ab/Ag
(Bio-Rad)

HIV-1 p24
Kivuyo Infants, DBS, ELISA Amplicor PCR
100 95.5 27
2011 [62] Tanzania (Perkin (Roche)
Elmer)

Determine 0.00 (acute) 98.3 (acute)

UniGold Recombigen
Ab/Ag 99.4 99.2
(Trinity Biotech),
(Inverness) (established) (established)
Bioline HIV 1/2 3.0 8 acute,
Adult >18
(Standard 163
yrs, whole HIV-1 p24
Rosenberg Diagnostics), HIV-1 established
blood, ELISA
2011 [28] p24 ELISA (Perkin ,
plasma, (Perkin
Elmer) with ELAST 838
Malawi Elmer) with 71.4 (acute) 100 (acute)
enhancement and negative
ELAST
Monitor HIV RNA
enhancemen
PCR (Roche)
t
 69 overall
(4x102-
5x104c/ml –
HIV p24
45, 5x104-
ELISA (NEN
1x105c/ml –
Adults, non- Life Science) 394
Spacek NAAT (Amplicor 62, 1x105-
B, plasma with ICD and 67 (331
2011 [63] PCR, Roche) 2.5x105c/ml –
Uganda ELAST patients)
68, 2.5x105-
enhancemen
5x105c/ml –
t
80,
>5x105c/ml –
90)

NAAT (PCR, HIV

NucliSENSEasyQ
Pregnant v2.0, bioMérieux),
Determine
Chetty women, Bioline HIV 1/2 3.0 Ag – 3.1, Ab
Ab/Ag 96.9 32
2012 [30] plasma, (Standard – 59.4
(Inverness)
South Africa Diagnostics), SENSA
Tri-line HIV-1/2/O
(Hitech)

Ag EIA (Beckman
Adults, acute
Coulter), Vironostika
infection, Determine
Kilembe HIV Uni-Form II Ag – 1.9, Ab
plasma, Ab/Ag 96.7 82
2012 [27] Ag/Ab, (bioMérieux), – 76.7
Rwanda/Zam (Inverness)
Capillus and UniGold
bia
(Trinity Biotech)

Architect HIV Genetic If Ab-ve:

Ag/Ab Systems NAAT
87.8
Combo HIV 1/2 + (Aptima
(Abbott) O (Bio- HIV-1 RNA
Rad) or Qualitative
Determine
Adults, acute Oraquick (Hologic) &
Patel 2012 HIV-1 Ag/Ab
infection, (OraSure Versant 75.8 ND 33
[35] Rapid Test
plasma, USA ) or HIV-1 RNA
(Alere)
OraQuick 3.0,
Genetic Advance (Siemens))
Systems HIV (OraSure , retest with
57.5
1/2 + O (Bio- )or HIV 1/2 +
Rad) Vironosti O (Genetic
 Multispot ka HIV-1 Systems)
HIV-1/HIV-2 MicroELI and
33.3
Rapid test SA Multispot
(Bio-Rad) (bioMérie HIV-1/HIV-
ux) 2 (Bio-
Clearview
Rad). If
Complete
Ab+ve:
HIV 1/2 29.6
Western
assay
blot
(ChemBio)
(manufactu

Unigold rer not

Recombigen stated)
24.2
HIV (Trinity
Biotech)

Clearview
HIV 1/2 Stat-
22.6
pak
(ChemBio)

OraQuick
Advance
Rapid HIV- 21.9
1/2 Ab
(OraSure)

Determine
Brauer Serum, Ab/Ag Ag – 10
Various*5 100 79
2013 [33] South Africa Combo Ab - 91
(Alere)

EIA (Architect HIV-

Acutely 1/2 Ag/Ab, Abbott), Ag – 29.4,
Faraoni infected Determine NAAT (CAP/CTM, Ab – 58.8,
100 17
2013 [29] adults, Ab/Ag (Alere) Roche), Western blot Ag or Ab –
serum, Italy (New LAV Blot, Bio- 88.2
Rad)

Elecsys HIV
Lysate
combi PT - 99.9 4465
supernatants
(Roche)
Tao 2013 (for Ag or
Various*6
[12] p24),
Elecsys HIV
seroconversi
combi - 99.9 675
on panels,
(Roche)
 NIBSC Advia
reference Centaur HIV
- 99.5 1039
panels, combo
plasma/seru (Siemens)
m, Korea,
Architect HIV
China, &
combo - 99.8 2751
Malaysia
(Abbott)

Vironostika
HIV Uni-
- 100.0 703
Form II Plus
O (Abbott)

Zhuhai
Livzon Anti-
HIV EIA
- 100.0 675
(Zhuhai
Livzon
Diagnostics)

Elecsys HIV 1.05 IU/mL -

Combi PT

Architect HIV 0.94 IU/mL -

Ag/Ab combo
(Abbott)
NIBSC Ag
standard Advia Not applicable 1.89 IU/mL - 6 dilutions
(90/636) Centaur HIV
Ag/Ab combo
(Siemens)
Mühlbacher
AxSYM HIV 1.20 IU/mL -
2012 [6]
Ag/Ab
(Abbott)

Blood donors Architect HIV Ag/Ab - 99.9 7343

combo (Abbott),
Routine - 99.8 4103
Advia Centaur HIV
screening
Elecsys HIV Ag/Ab combo
samples
Combi PT (Siemens), AxSYM
Cross- HIV Ag/Ab (Abbott), - 99.3 296
reactivity Prism HIV O Plus
samples (Abbott)
 Architect HIV Ag/Ab
Combo (Abbott),
Adults, BioPlex 2200
Salmona ImmunoComb II HIV-
serum, Ag-Ab (Bio- 100 99.4 1505
2014 [23] 1 & 2 BiSpot
France Rad)
(Orgenics), New Lav
Blot I & II (Bio-Rad)

AxSYM HIV
1/2 gO 100 100
(Abbott)

HIV (1+2)
Ag/Ab
(Beijing New LAV Blot I (Bio- 98.8 100
Chang Adults, Wantai Bio- Rad), Cobas
152
2015 [9] plasma pharm) Amplicor HIV-1
Monitor v1.5 (Roche)
HIV Combi
Ag/Ab EIA 100 98.5
(Bio-Rad)

HIV Ab/Ag
100 100
(Dia.Pro)

Determine HIV-1/2
(Alere), SD Bioline
HIV 1/2 v3 (Standard
GS HIV
Piwowar- >16 years, Diagnostics), Established –
Combo
Manning plasma, UniGold HIV (Trinity 100 96.7 612
Ag/Ab EIA
2015 [21] whole blood Biotech), Architect Acute – 83.3
(Bio-Rad)
HIV Ag/Ab (Abbott),
Aptima HIV-1 RNA
(Hologic Gen-Probe)

Enzygnost
HIV Integral
100 100
II Ag/Ab
Inno-Lia HIV I/II
ELISA
Adults, immunoblot
Urio 2015
serum, Murex HIV (Innogenetics, 600
[19] 100 100
Tanzania Ag/Ab Belgium)

Vironostika
HIV Uniform 100 99.5
II Ag/Ab
 VL
ELISA +
<3,000c/mL:
Bystryak Children, photochemic
Amplicor HIV-1 Monitor 52.6
2016 plasma/ serum, al signal 100 182
(Roche) VL
[64] USA amplification
>3,000c/m L:
system
100

Blood donor Western Blot (details

xMAP
Kong 2016 panels (early not specified), NAAT
(Luminex, 57.8 - 33
[65] infection), (RNA, details not
USA)
China specified)

NAAT (Aptima HIV-1

Architect
Peters 2016 Adults, acute RNA, Gen-Probe, or
Ag/Ab 79.8 99.9 168
[14] infection, USA m2000 RealTime HIV-
(Abbott)
1, Abbott)

OraQuick
Advance
RNA+ve: 75.0
HIV-1/2 99.9 2180
Ag+ve: 85.0
(OraSure),
saliva

OraQuick
Advance
RNA+ve: 77.9
HIV-1/2 100.0 2175
Ag+ve: 88.3
(Orasure),
whole blood
Adults, acute
Uni-Gold Pooled NAAT
infection, whole RNA+ve: 84.9
Stekler 2016 (Trinity (RealTime HIV-1 RNA, 100.0 1614
blood/ serum/ Ag+ve: 95.7
[66] Biotech) Abbott), Architect HIV-
plasma/ saliva
1 Ag/Ab (Abbott)
USA
INSTI HIV-1 RNA+ve: 73.3
99.8 559
(bioLytical) Ag+ve: 84.6

Determine
Ag/Ab RNA+ve: 84.6
99.0 1523
Combo Ag+ve: 91.7
(Alere)

GenScreen
HIV-1/HIV-
RNA+ve: 87.9 99.8 2161
2+O Ab EIA
(Bio-Rad)
 Elecsys HIV
Combi PT VIDAS Duo EIA 100 99.7
(Roche) (bioMérieux), Inno-Lia
Blaich 2017 Adults, serum, HIV I/II (Innogenetics),
3997
[67] Switzerland Architect HIV NAAT (Cobas
Ag/Ab AmpliPrep/Cobas
100 99.8
Combo TaqMan HIV-1, Roche)
(Abbott)

Stored and
HIV-1/2 Architect HIV Ag/Ab
fresh samples,
Fitzgerald Ag/Ab Combo (Abbott), p24: 88
plasma/ serum/ 100 120
2017 [68] Combo VIDAS Duo EIA Ab: 100
whole blood,
(Alere) (bioMérieux)
UK

Architect HIV Ag/Ab

Combo (Abbott),
Liason XL Western blot (New LAV p24:
Ghisetti 2017 Adults/panels,
HIV Ag/Ab Blot, Bio-Rad), NAAT 9.9pg/mL 99.7 3090
[69] Italy
(DiaSorin) (Cobas Ab: 100
AmpliPrep/Cobas
TaqMan HIV-1, Roche)

Determine Architect HIV Ag/Ab

Masciotra
Adults, stored HIV-1/2 Combo (Abbott), p24: 50.0
2017 100 508
plasma, USA Ag/Ab MultiSpot HIV-1/HIV-2 Ab: 99.6
[70]
Combo Rapid (Bio-Rad)

Cobas
Infants, whole AmpliPrep/Cobas
Meggi 2017 Lynx p24 Ag
blood, TaqMan HIV-1 71.9 99.6 879
[71] POC
Mozambique (Roche), Amplicor HIV-
1 Monitor (Roche)

Determine
Advia Centaur HIV-
HIV-1/2
1/O/2 (Siemens), HIV-1 95 100 133
Combo
Western blot, Aptima
(Alere)
HIV-1 RNA (Hologic),
Stafylis 2017 Stored serum,
Architect HIV Ag/Ab
[72] USA SD Bioline
Combo (Abbott),
HIV Ag/Ab
Cobas
Combo 91 100 133
AmpliPrep/Cobas
(Standard
TaqMan HIV-1 (Roche)
Diagnostics)
 Acute (p24):
Architect HIV Ag/Ab
65
Combo (Abbott),
HIV-1/2 Recent
van Tienen Adults, stored Liason XL HIV Ag/Ab
Ag/Ab (p24): 24
2017 serum, (DiaSorin), VIDAS Duo - 89
Combo Recent (Ab):
[73] Netherlands EIA (bioMérieux), Inno-
(Alere) 100
Lia HIV Ab
Chronic (Ab):
(Inogenetics)
100

Enzygnost
Anti-HIV 1/2 11.4 100
Plus

Vironostika
HIV Ag/Ab 34.3 95
(bioMérieux)

Innotest HIV
Ag mAb EIA 45.7 100
(Fujirebio)

Determine
Inno-Lia Score I/II
HIV-1/2 6.1 -
(Innogenetics),
(Alere)
Seroconverters neutralization by
Fransen 2017 (acute), Innotest HIV Ag mAb
Determine 77
[74] plasma, (Fujirebio), NAAT
HIV-1/2 8.6 93.1
Belgium (Cobas
Ag/Ab (Alere)
AmpliPrep/Cobas
SD Bioline TaqMan HIV-1, Roche)
HIV-1/2
12.1 -
(Standard
Diagnostics)

SD Bioline
HIV Ag/Ab
Combo 29.4 100
(Standard
Diagnostics)

Alere HIV
17.1 93.1
Combo

Eshleman Adults, acute Architect HIV Oraquick Advance HIV-

45.8 - 24
2018 [75] infection, Ag/Ab 1/2 Ab (OraSure),
 plasma, Combo UniGold Recombigen
USA/South (Abbott) (Trinity Biotech),
Africa Geenius HIV-1/2 Ab
GS HIV
(Bio-Rad), NAAT
Combo
(Aptima HIV-1, 50.0
Ag/Ab EIA
Hologic)
(Bio-Rad)

BioPlex 2200
HIV Ag-Ab 45.8
(Bio-Rad)

Lumipulse
Western blot (HIV blot
HIV Ag/Ab 100 99.2
Adults/panels, 2.2, MP Diagnostics),
Zhao 2018 (Fujirebio)
serum/lysate, NAAT (Cobas 1153
[76]
China AmpliPrep/Cobas
Elecsys HIV
TaqMan HIV-1, Roche) 100 100
Combi PT

Supplementary Table 3. Reported sensitivity and specificity of p24 antigen detection from selected studies. Studies
were selected that were designed to comprehensively measure one or more index assay characteristics (e.g.
sensitivity, specificity, subtype-breadth) against one or more reference methods.
§
Sensitivity and specificity, where given, are for the Ag detection part of the test unless otherwise stated. Names of
companies or assay manufacturers, as far as possible, are given as those at the time of print of the original paper.
ELAST enhancement; a tyramide signal amplification system that improves sensitivity for standard horseradish
peroxidase-based ELISAs
Abbreviations. Ag: p24 antigen; Ab: antibody; DBS: dried blood spot; ICD: immune-complex disruption; IQR: inter-
quartile range; N/A: not applicable; NAAT: nucleic acid amplification test; PBMC: Peripheral blood mononuclear cells;
TMA: transcription-mediated amplification.
*1 Two modifications were proposed to the manufacturers guidelines for the assay; the best results of the two
modifications are presented.
*2 HIV-1/HIV-2 3rd gen Plus EIA (Genetic Systems), IMx HIV-1/HIV-2 III Plus (Abbott), AxSYM HIV-1/2 gO (Abbott),
Prism HIV-O Plus (Abbott), VIDAS HIV DUO (bioMérieux), Genscreen HIV-1/2 (Bio-Rad), Enzygnost Anti-HIV1/2
Plus (Dade Behring), Enzygnost HIV Integral (Dade Behring), Enzygnost Anti- HIV (Dade Behring), Enzymun-Test
HIV Combi (Boehringer), and Cobas Core Anti-HIV 1 2 O EIA (Roche Diagnostics).
*3 AxSYM HIV Ag/Ab Combo (Abbott), Enzygnost HIV Integral (Dade Behring), Genscreen Plus HIV Ag/Ab (Bio-
Rad), Murex HIV Ag/Ab Combo (Abbott), VIDAS HIV DUO (bioMérieux) and Vironostika HIV Uniform II Ag/Ab
(Organon Teknika).
*4 Vironostika HIV Uni-Form (Organon Teknika), HIV1/HIV2 ELISA kit (Cambridge Biotech), Vironostika HIV Uniform
II plus O (bioMérieux), the Vironostika HIV Uni-Form II Ag/Ab (bioMérieux) and/or the Enzygnost Anti-HIV 1/2 Plus
test (Dade Behring). Reactive samples were further characterized with the Inno-Lia HIV Confirmation assay or the
Inno-Lia HIV I/II Score test (Innogenetics)
*5 Cobas HIV Combi kit on Modular E170 (Roche Diagnostics), HIV Ag/Ab Combo kit on AxSYM (Abbott) and HIV
Ag/Ab Combo kit on Architect i2000 (Abbott). Two of the HIV-1/2 antibody reactive specimens had confirmatory
testing performed on the Determine HIV-1/2 assay (third generation rapid assay) instead of a fourth-generation HIV-
1/2 ELISA. The Cobas HIV Ag kit (Roche) on Modular E170 was used for initial p24 antigen determination on the
diagnostic specimens.
*6 Advia Centaur HIV Ag/Ab combo (Siemens), Architect HIV Ag/Ab combo (Abbott), Vironostika HIV Uni-Form II
Plus O (bioMérieux), Elecsys HIV combi (Roche),
 Zhuhai Livzon Anti-HIV EIA (Zhuhai Livzon Diagnostics), Serodia
HIV1/2 Particle Agglutination (Fujirebio).
Supplementary Table 4
Method
Reference Treatment
described
Kageyama 1988 200 µl sample mixed with 200 µl glycine-HCl pH 2.0, heated to 70C for
Heat/Acid
[77] 5/10 min, neutralized with 3 µl 5 M Tris; Tested by Abbott Ag EIA
1 M HCl added to 400 µl serum to give pH 3.0 (usually 30 µl), incubated at
Mathiesen 1988
Acid RT for 90 min, then 4C, neutralized to pH 7.4 with 1 M NaOH, immediately
[78]
used for ELISA
100 µl serum mixed with 25 µl 0.5 M HCl, incubated RT 90 min then
Von Sydow 1988
Acid neutralized 25 µl 0.5 M NaOH and diluted to 200 µl with PBS. Used in
[79]
ELISA
100 µl serum mixed with 50 µl 0.5 N HCl pH 2.5-3.0, incubated 60 min
Nishanian 1990 [80] 37C, neutralized with 50 µl 0.5 N NaOH to pH 7.0. 22 µl Triton X-100
added, then 200µl used for ELISA
ELISA: 200 µl 1.5 M glycine pH 1.85 added to 200 µl serum, mix, incubate
Ascher 1992 [81] Acid
37C 60 min, neutralize 1.5 M Tris pH 9.0
100 µl specimen mixed 0.5 N HCl, 50 µl incubated 60 min 37C, neutralized
Bollinger 1992 [82] Acid
50 µl 0.5 N NaOH, then ELISA
100 µl plasma or serum mixed with 200 µl glycine-HCl pH 2.0, heated to
70C 10 min. Neutralized to pH 7.4 with 10-20 µl 5 M Tris
Papaevangelou OR
Acid or Heat
1992 [83] 200 µl plasma or serum titrated with 1 M HCl (usually ~20µl) to pH 3.0,
incubated RT for 90 min, then placed on ice and neutralized to pH 7.4 with
1 M NaOH (usually 20 µl)
100 µl plasma mixed with 190 µl 0.15 M glycine-HCl pH 2.0. Heated 70C
10 min, neutralized by 10 µl 5M Tris. 200 µl used for ELISA
Chandwani 1993
2x Acid OR
[37]
100 µl plasma mixed with 50 µl 0.5N HCl (pH 2-3), incubated 37C 60min,
neutralized 50 µl 0.5 N NaOH on ice
100 µl serum mixed 100 µl glycine pH 2.5 37C 90min, then neutralized
Fenouillet 1993 [84] Acid
with 100 µl Tris pH 7.5.
PEG: 450 µl serum incubated with 350 µl 0.2 M EDTA and 200 µl of 12%
PEG for 16-20 h at 4C. Mix spun at 8,000 xg for 15 min at 4C. S/n
frozen at -70C; pellet washed 6x in 20x vol of cold PBS, then resuspended
Kashala 1993 [85] PEG or Acid in 6 M guanidinium HCl
Acid: 100 µl serum incubated with 0.5 N HCl pH 3 1 h 37C, then
neutralized with 0.5 N NaOH. Triton X-100 added, and sample assayed
Note: Abs released from disruption also assayed
Coulter protocol: 1:1 dilution of serum with 1.5 M glycine-HCl pH 1.8,
Lillo 1993 [86] Acid/Heat
incubated 90 min 37C then neutralized with 1.5 M Tris pH 9.0
 Coulter ICD kit: 70 µl sample added to 70 µl 1.5 M glycine-HCl pH 1.8,
Miles 1993 [87] Acid/Heat 37C 90 min, then 70 µl 1.5 M Tris-HCl pH 7.4, incubate 2 h. 200 µl used
for ELISA
After [80]. 200 µl serum mixed with 100 µl 0.5 N HCl then 37C 60 min.
Pokriefka 1993 [88] Heat/Acid
Adjusted to pH 7 with 0.5 N NaOH, assayed by ELISA
Quinn 1993 [38] Acid Not described, but [80] and [82] referenced
Schüpbach 1993 Serum or plasma diluted 1:3 with distilled H2O or 0.5% Triton X-100, ‘boiled
Heat
[89] in dry heat block’ (100C for 2, 3 or 5 min)
Coulter ICD-prep kit. 10 µl lysis reagent, 100 µl sample and 100 µl 1.5 M
Simon 1993 [90] Acid glycine-HCl pH 1.85 mixed, incubated 37C for 90 min then 100 µl 1.5 M
Tris pH 9 added
100 µl serum added to 1.5 M glycine pH 1.8-2.2, incubated 37C 1 h, then
neutralized with 100 µl 1.5 M Tris pH 8.6-9.0
Vasudevachari
Acid or Heat OR
1993 [91]
100 µl serum incubated 1 h 50 µl 0.5 N HCl, then neutralized with 50 µl 0.5
N NaOH
Duiculescu 1994 100 µl plasma mixed 100 µl 1.5 M glycine-HCl pH 1.85, incubated 37C for
Acid
[92] 90 min, then neutralized with 100 µl 1.5 M Tris-HCl; 200 µl used for ELISA
Morand-Joubert 100 µl serum added to 190 µl 0.15 M glycine-HCl pH 2, incubated at 70C
Acid
1994 [93] for 10 min, neutralized with 10 µl 3.5 M Tris
Schüpbach 1994
Heat Samples diluted with 2 volumes of 0.5% Triton X-100, boiled for 5min
[94]
Brown 1995 [95] Acid ICD-Prep kit, Coulter Immunology
100 µl plasma incubated 100 µl glycine and 30 µl lysis buffer, 90 min, 37C
Bulterys 1995 [40] Acid
then neutralized with 100 µl Tris. 200 µl then used for ELISA
Coulter ICD; 300 µl serum, 150 µl 1.5 M glycine-HCl, incubated 37C for 60
Gutierrez 1995 [96] Acid
min, neutralized with 1.5 M Tris. 200 µl used for ELISA
Kappes 1995 [97] Acid Coulter assay, details not given, but see [98]
Coulter ICD-prep kit: 100 µl plasma added to 100 µl 0.15 M glycine-HCl
Lewis 1995 [41] Acid/Heat with Triton X-100, incubated 90 min 37C, neutralized with Tris. 200 µl
used for ELISA
100 µl plasma added to 190 µl glycine-HCl, then 10 min at 70C, then 10 µl
Nielsen 1995 [42] Acid/Heat
Tris base. 200 µl used for ELISA
Coulter ICD kit;100 µl sample incubated with lysis buffer and glycine for 90
Guay 1996 [99] Acid
min, neutralized with Tris, then 200 µl used for ELISA
Samples diluted (1:3, 1:6 or a few 1:12/1:24) with 0.5% Triton X-100, boiled
Lyamuya 1996 [43] Heat
for 5 min at 100C. 250 µl used for ELISA
Stanojevic 1996 100 µl serum incubated with 50 µl 0.5 M HCl for 60 min 37C, then 50 µl
Acid
[100] 0.5 M NaOH. Analysed by ELISA
100 µl plasma diluted 500 µl 0.5% Triton X-100, 5 min at 100C, then
Boni 1997 [101] Heat
ELISA
 70 µl plasma added to 70 µl 1 M glycine buffer pH 1.85, 21 µl 5% Triton X-
Nesheim 1997 [44] Acid 100. Incubated 37C 1 h, then 70 µl 1M Tris pH 9.0. 200 µl used for ELISA
(Coulter)
Panakitsuwan 1997 Acid – Coulter ICD-Prep kit
Acid or Heat
[45] Heat – following method of [94]
Samples added to 22.5 µl lysis buffer, then 75 µl glycine, then incubated
Paul 1997 [46] Acid 37C 90 min. Then 75 µl Tris, and 200 µl each mixture added to ELISA
reaction plate
Rich 1997 [47] Acid Coulter ICD_Prep kit, according to manufacturer’s instructions
Fackler 1998 [102] Acid Not described: Coulter ELISA
Heat - Plasma diluted 1/3 with 7mM SDS/1.5mM DTPA pH 7.2, incubated
Comparison
95-98C 4min
of methods
Steindl 1998 [103] Heat – Plasma diluted 1/3 with distilled H2O, incubated 100C 5min
on model
Acid – Plasma diluted 1/3 with 1.5 M glycine-HCl pH 1.85, incubated 37C
samples
1 h, then neutralized 100 µl 1.5 M Tris-HCl pH 9.0 to 200 µl
100 µl plasma diluted with 500 µl 0.5% Triton X-100, heated 100C 5 min,
Nadal 1999 [104] Heat
tested by ELISA
Ortigão-de-Sampaio
Acid Glycine-HCl (no further details)
1999 [105]
Ledergerber 2000
Heat 100 µl plasma diluted with 500 µl 0.5% Triton X-100, 5 min 100C.
[106]
Read 2000 [107] Acid Detail not given, but see [98]
Sutthent 2003 [50] Heat Plasma diluted 1:6 with 0.5% Triton X-100, heated 100C for 5 min
Plasma diluted 1:6 in 0.5% Triton X-100, heated 100C 5 min. 250 µl used
Prado 2004 [108] Heat
for ELISA
25 µl plasma mixed with 75 µl (0.67%NP-40 0.2% SDS in PBS), heated 4
Parpia 2010 [109] Heat
min 88C, then cooled and assayed by dipstick
Supplementary Table 4. Methods explored to disrupt immune complexes of host antibody and p24 in blood, prior to
diagnostic analysis. A brief summary of the method, where described, is given.
Supplementary Table 5

Assay Limit of Detection Development stage References

Immune complex transfer enzyme

26 fg/mL No known further development [110-112]
immunoassay

<1 virion* or
Immuno-PCR No known further development [113,114]
0.2 fg/mL

Radioimmunoassay 50 fg/mL No known further development [115]

Magnetic immunochromatography 17-33pg/mL No known further development [116]

Lateral flow with fluorescence <1 pg/mL No known further development [117]

Lateral flow dipstick 50 pg/mL Under commercialisation [109]

Europium nanoparticle–based 0.5 pg/mL

immunoassay, microchip and 0.1 pg/mL Ongoing, at research stage [118-121]
biobarcode 5 pg/mL

50 pg/mL
Amperometric immunosensor 8 pg/mL No known further development [122-124]
0.5pg/mL

Capacitive immunosensor. 0.25 fg/mL Ongoing, at research stage [125,126]

Amperometric immunosensor 6.4 pg/mL No known further development [127]

Ongoing, at research stage.

Immunoliposome PCR 0.24 fg/mL [128]
Patent WO2005067583 A2

Plasmonic ELISA 1 ag/mL Ongoing, at research stage [129,130]

4.9 fg/mL Commercially available

Digital immunoassay [131-133]
2.5 pg/mL (Quanterix Corp.)

Electrochemiluminescence sandw
1 pg/mL TBD [134]
ich immunosensor

Fluorescent protein array 54 pg/mL No further development [135]

Rapid immunofiltration assay 420 pg/mL Under commercialisation [136]

Nanoribbon field-effect transistor

20 fg/mL TBD [137]
biosensors

Thio-NAD cycling amplified ELISA 25ag/mL TBD [138]

 Photochemical signal Under commercialization,
80 fg/mL [64,139]
amplification system for ELISA Patent US8916341 B1

Carbon-dot microfluidic
20pg/mL TBD [140]
immunoassay

Carbon-dot paper ELISA 250pg/mL TBD [141]

Zinc nanowire origami biosensor 300fg/mL TBD [142]

Colloidal Gold
25pg/mL TBD [143]
Immunochromatographic Assay

3.7-30,000pg/mL
Cytometry bead assay TBD [144]
(subtype dependent)

Birefringence ELISA 250pg/mL TBD. Patent WO2015185504A1 [145]

Magnetic bead ELISA 0.5pg/mL TBD [146]

Cantilever with optoplasmonic

0.01-0.5fg/mL TBD [147]
transduction

Platinum nanocatalyst lateral flow 0.8pg/mL TBD [148]

Carbon nanotube/ imprinted

polymer-based electrochemical 83fg/mL TBD [149]
sensor

ELISA with peroxide strip readout 11.6pg/mL TBD [150]

Dendrimer nanochain lateral flow 5ng/mL TBD [151]

Surface acoustic wave biosensor 48ng/mL TBD [152]

Supplementary Table 5: Selected ultrasensitive assays for p24 antigen. fg, femtogram; ag, attogram. Sensitivities
or limits of detection given are those calculated by study authors and have not been verified independently. Studies
also used a variety of samples including biological buffers or healthy volunteer samples spiked with recombinant p24,
or actual patient samples. *Sample volume not reported, TBD, to be determined (for papers from 2014 onwards).
Corresponding authors for all papers prior to 2016 were emailed in January 2017 requesting a status update for the
studies listed. In the case of invalid email addresses, contact with another author was attempted.
Supplementary Table 6

Biomarker Observed References Summary of findings and study notes

relationship
with p24

Viral load Yes Read 2000 [107] ICD p24 levels correlated with other biomarker levels
(anti-p24 antibody, CD4% and RNA).

Pascual 2002 [153] HIV RNA and heat dissociated p24 assay were
significantly correlated (R2=0.6, p<0.0001). The modified
p24 assay with heat dissociation step was more sensitive
than the unmodified assay.

Fiebig 2003 [154] Concurrent increase of HIV-1 RNA and p24 antigen
during acute stage of the disease (pre-seroconversion).
Slopes (from linear regression of log transformed data)
were strongly correlated (R2 = 0.82).

Ribas 2003 [155] Correlation with RNA (Spearman -test, R = 0.751,

p<0.0001). p24 assay detected a wider range of
subtypes than the viral load assay. p24 outside viral
particles has a half-life of 42 days, hence stays
temporarily detectable even after viral loads decrease.

Prado 2004 [108] Anti-p24 antibody levels have key influence on detection
of p24. A significant correlation between HIV RNA and
p24 antigen assays was found (p<0.001,  = 0.23; logistic
regression), but this was weaker and a more gradual
slope for those on a structured treatment interruptions
program. p24 assays are not sensitive enough for
monitoring in some patients with high CD4 counts.

Stevens 2005 [156] Subtype C samples. Moderate correlation between log10

RNA viral load. Major concern is variability and lack of
sensitivity.

Brinkhof 2006 [157] Evaluated p24 for treatment monitoring in children, and in
relation to CD4 levels. Correlation between p24 and RNA
(p<0.0001). Statistical support for changes in p24 and
RNA with respect to CD4 levels. Study of children.

Erikstrup 2008 [158] p24 correlated with RNA level (p<0.0001, R2 = 0.44).

No Coombs 1989 [159] p24 not as good as plasma viraemia culture counts. No
correlation between RNA and p24 or antibody and
p24. No immune complex disruption step.
 Unclear Brown 1995 [160] Antibody level is important and discordant results occur:
(i) p24 can be higher than RNA due to circulating non-
virion associated p24 and (ii) antibodies may mask p24.

Respess 2005 [52] Correlation with RNA level, though not for viral loads of
<5,000c/ml. p24 detection is suitable for paediatric
diagnoses, where viral loads are high.

Disease Yes Spector 1989 [161] Used p24 antigen as a virological marker during a trial of
Progression zidovudine and ribavirin treatment. Decline in
antigenaemia can be used as a biomarker in trials of
drugs.

MacDonnell 1990 [162] Detectable p24 antigenaemia was strongly associated

with more rapid progression to AIDS, regardless of initial
CD4 cell count.

Katzenstein 1992 [163] Plasma p24 level correlated with 2-tier stage of disease
(p<0.001 for disease stage)

Morand-Joubert 1994 p24 is an earlier marker of disease progression when

[93] used with an immune complex disruption step.

Bulterys 1995 [40] Detection of high levels of p24 strongly linked to rapid
progression to AIDS and earlier death in babies. Children
with lower p24 (<50pg/mL) survived longer (20 months vs
7 months, p-0.02).

Farzadegan 1996 [164] Rapid disease progression was more common in people
with detectable p24.

Ledergerber 2000 [106] Mean p24 increased gradually from early to late stage of
the disease. p24 protein level was a significant
prognostic factor of survival: RNA (p<0.005) and p24
(p=0.043) both predictors of progression to AIDS.

Read 2000 [107] ICD p24 antigen levels were correlated with disease
progression better than, or equivalent to, HIV RNA levels
or CD4 percent.

Sterling 2002 [165] p24 predicted disease progression in early stage HIV
especially when combined with information on CD4+
lymphocyte count and HIV viral load. p24 tests (even
quantitative tests) were cheaper than viral load or CD4
counts.
 Erikstrup 2008 [158] p24 was a better predictor of Center for Disease Control
category than CD4 count (p<0.001) but a worse predictor
of mortality than HIV-RNA and CD4 count.

Unclear Baillou 1987 [166] Variation seen in correlation of p24 antigenaemia with
progression to AIDS in different populations. In
Europeans, persistent detectable antigen associated with
transition to AIDS. In Central Africans, detectable antigen
was not found in patients with AIDS.

Duiculescu 1994 [92] p24 level decreased during zidovudine treatment. Rapid
progression of disease associated with unchanged levels
of p24 after treatment. Better information obtained by
considering the results from p24 with and without ICD.

Schüpbach 2005 [167] Studied p24 dynamics during structured treatment

interruptions. p24 responded less rapidly to breaks in
HAART than viral load. p24 increases, but not viral load
during the first 8 weeks, was inversely correlated to
changes in CD4 levels. p24 levels did not always reduce
during treatment that successfully reduced viral load.

CD4 Count Yes Katzenstein 1992 [163] Plasma p24 level correlated with CD4 count (p<0.05 for
CD4 cell count).

Duiculescu 1994 [92] Lower CD4 percent found in those who test positive for
p24 antigen levels compared to those who test negative.
Study of children.

Ledergerber 2000 [106] p24 was found to be a better or equivalent predictor of

CD4 depletion than RNA.

Read 2000 [107] ICD p24 levels correlated with other biomarker levels
(anti-p24 antibody, CD4% and RNA).

Schüpbach 2005 [167] Studied p24 dynamics during structured treatment

interruptions. p24 responded less rapidly to breaks in
HAART than viral load. p24 increases, but not viral load
during the first 8 weeks, was inversely correlated to
changes in CD4 levels. p24 levels did not always reduce
during treatment that successfully reduced viral load.

Stevens 2005 [156] Subtype C samples. Highly significant (inverse)

correlation between CD4 and p24 level (p<0.0001).
Major concern is variability and lack of sensitivity.
 Brinkhof 2006 [157] Evaluated p24 for treatment monitoring in children, and in
relation to CD4 levels. Statistical support for changes in
p24 and with respect to CD4 levels. Study of children.

Anti-p24 Yes Duiculescu 1994 [92] Anti-p24 antibody level was inversely correlated with p24
Antibody antigen.

Read 2000 [107] ICD p24 levels correlated with other biomarker levels
(anti-p24 antibody, CD4% and RNA).

Supplementary Table 6. Summary of studies investigating the relationship between quantitative p24 levels, other
biomarkers, and disease progression.
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